Figure 1.

The ketone body βHB sustains spontaneous SNr firing in the absence of glucose, but glycolytic inhibition slows the firing rate. A, SNr spontaneous firing rate in the presence of glucose (G; 10 mm; 31.4 ± 3.2 spikes/s; black symbols, n = 10). Spontaneous firing rate (29.3 ± 4.5 spikes/s; blue symbols; n = 8) recorded from neurons in the absence of glucose but in the presence of βHB (2.5 mm) for at least 20 min was not significantly different from the firing rate in glucose (p > 0.05, one-way ANOVA with Bonferroni's test). In the absence of external fuel (0 mm glucose; 0 mm βHB), SNr spontaneous firing was almost completely silent (0.9 ± 0.4 spikes/s; red symbols; n = 10). B, When glycolysis was inhibited using 2-DG (5 mm) in the absence of glucose and βHB (black line), the spontaneous firing of an SNr neuron decreased and then sharply dropped until firing was silenced. Return of glucose in the external solution rapidly restored the spontaneous firing. In the presence of βHB (2.5 mm; blue line), glycolytic inhibition with 2-DG (5 mm) in the absence of glucose decreased the spontaneous firing of a SNr neuron but did not silence it. C, When glycolysis was inhibited with IAA (1 mm) in glucose solution (10 mm; black trace), the firing rate of a representative SNr neuron decreased and then sharply stoped firing action potentials. However, in the presence of the ketone body βHB (2.5 mm; blue trace) and absence of glucose, the firing of a representative SNr neuron was sustained after glycolytic inhibition with IAA at a lower firing rate. D, Average firing rate of SNr neurons in control (Ctrl) and test conditions. The initial firing rate of SNr neurons in glucose (10 mm) was 32.7 ± 2.9 spikes/s (black squares and line; n = 12), but after removal of glucose and addition of 2-DG (5 mm), the firing rate was almost completely silenced within 15 min (0.8 ± 0.3 spikes/s). The firing rate of SNr neurons in the absence of glucose but in the presence of βHB (2.5 mm) was 33.1 ± 3.7 spikes/s (blue square and line). After addition of 2-DG (5 mm or 10 mm), the firing rate of those SNr neurons was significantly reduced but not silenced (22.2 ± 2.6 spikes/s; n = 13; p = 0.0001, Student's paired t test). Addition of IAA (1 mm) to spontaneously firing SNr neurons (37.7 ± 9.7 spikes/s; n = 6; black circles and line) in glucose (10 mm) completely silenced SNr firing. When exogenous βHB (2.5 mm; blue circles and line) replaced glucose, the spontaneous firing of SNr neurons (37.2 ± 2.7 spikes/s) was significantly decreased after addition of IAA (1 mm) but was not silenced (17.8 ± 1.4 spikes/s; n = 20; p = 4.8 × 10⁻⁷, Student's paired t test). Similarly, when lactate (5 mm; n = 5; red circles and line) replaced glucose, SNr spontaneous firing (39.8 ± 4.5 spikes/s) was decreased after addition of IAA (21.6 ± 3.4 spikes/s; p = 0.002, Student's paired t test). The mitochondrial poisons rotenone (Rot; 1 μm) and oligomycin (Oligo; 1 μm) silenced SNr firing in glucose solution (33.7 ± 3.0 vs 0.8 ± 0.5 spikes/s; n = 6; black diamonds and line). In the presence of rotenone (1 μm) and oligomycin (1 μm), βHB (2.5 mm) did not sustain SNr firing after treatment with IAA (40.2 ± 7.4 vs 0.2 ± 0.2 spikes/s; n = 4; blue diamonds and line). E, Replacement of glucose with 2-DG and βHB decreased SNr firing by 32.3 ± 5.0% (n = 13), which was significantly less than the decrease observed when using IAA to inhibit glycolysis (p < 0.05, one-way ANOVA with Bonferroni's test). Inhibition of glycolysis with IAA was performed with βHB either in the absence of glucose (blue symbols) or with 10 mm glucose (red symbols). The percentage decrease in firing rate was not significantly different (p > 0.05, one-way ANOVA with Bonferroni's test) between experiments without glucose (49.5 ± 3.4%; n = 20) or while maintaining glucose (64.2 ± 5.8%; n = 12). In the presence of the antioxidant Tempol (2 mm), the percentage decrease in firing rate (64.2 ± 5.2%; n = 8) after inhibition of glycolysis with IAA in the presence of βHB and glucose was not significantly different from control experiments without Tempol. All error bars indicate SEM; *p < 0.05.